Loss of full-length hnRNP R isoform impairs DNA damage response in motoneurons by inhibiting Yb1 recruitment to chromatin

Figure 1.. The Hnrnpr gene encodes two hnRNP R protein isoforms generated by alternative splicing. (A) Western blot analysis of hnRNP R isoforms using a C-terminal-specific antibody performed on tissue lysates from P5 wildtype mice. Equal protein loading in individual lanes was verified by probing with an antibody against β-Actin. (B) Western blot analysis of hnRNP R on brain lysates prepared from wildtype mice of different ages using the C-terminal-specific hnRNP R antibody. Equal protein loading in individual lanes was verified by probing with an antibody against Gapdh. (C) Schematic representation of the murine Hnrnpr locus that gives rise to a full-length Hnrnpr-FL mRNA and an exon 2-skipped Hnrnpr-ΔN mRNA produced by alternative splicing. Full‐length hnRNP R-FL and N‐terminally truncated hnRNP R-ΔN isoforms are shown. Protein domains are depicted as colored boxes: RRM (RNA-Recognition Motif), NLS (Nuclear Localization Signal), RGG (Arg-Gly-Gly box), and Q/N (Gln/Asn-rich domain). Constitutive exons are represented by gray boxes. Exon 2 (red box) is alternatively spliced. Introns are represented by horizontal lines. (D) Western blot analysis of lysates from HEK293TN cells transfected…

Figure 2.. A mouse model for knockout of the full-length isoform of Hnrnpr. (A) Schematic representation of the targeting strategy for disruption of the Hnrnpr locus. A gene-trap cassette containing a splice acceptor (SA) from the engrailed-2 (En2) gene, an internal ribosome entry site (IRES) directing the translation of a reporter (LacZ) and a SV40 polyadenylation sequence (pA) was inserted upstream of Hnrnpr exon 3. In Hnrnprtm1a/tm1a mice, splicing of exon 1 or exon 2 to En2 generates transcripts that terminate at the SV40 pA site (Transcripts 1 and 2). Utilization of a cryptic 5′ splice site in En2 results in a stable transcript (Transcript 3) with insertion of 115 nucleotides of En2 between exons 2 and 3 of Hnrnpr-ΔN mRNA. (B) qPCR analysis of total Hnrnpr (Hnrnpr-FL + Hnrnpr-ΔN) levels using primers specific to the 3′ UTR in brains of P5 Hnrnpr+/+,Hnrnprtm1a/+ and Hnrnprtm1a/tm1a mice. Data are mean ± SD (n = 4 animals). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test; n.s., not significant. (C) Western blot analysis of brain lysates obtained from P5 Hnrnpr+/+, Hnrnprtm1a/+ and Hnrnprtm1a/tm1a mice using an antibody against the…

Figure 3.. Depletion of full-length hnRNP R results in an up-regulation of the hnRNP R-ΔN isoform. (A) Absolute mRNA copy numbers of wildtype and chimeric Hnrnpr isoforms normalized to copies of Gapdh in brains of Hnrnpr+/+,Hnrnprtm1a/+ and Hnrnprtm1a/tm1a mice. Data are mean ± SD (n = 4 animals). (B) qPCR analysis of Hnrnpr pre-mRNA levels using primers specific to intron 1 in brains of Hnrnpr+/+, Hnrnprtm1a/+ and Hnrnprtm1a/tm1a mice. Data are mean ± SD (n = 4 animals). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test; n.s., not significant. (C, D) qPCR analysis of Hnrnpr-FL (C) or Hnrnpr-ΔN (D) levels in primary motoneurons transduced with an shRNA against exon 3 for depletion of both hnRNP R isoforms (shRNA-FL+ΔN), or with different shRNAs against exon 2 for depletion of the full-length hnRNP R isoform (shRNA-FL-A, shRNA-FL-B and shRNA-FL-C). Absolute copy numbers of Hnrnpr-FL (C) and Hnrnpr-ΔN (D) mRNA isoforms were normalized to absolute copies of Gapdh, controls were set to 1. Data are mean ± SD (n = 4 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test; *P ≤…

Figure 4.. The hnRNP R-ΔN isoform is sufficient to support axon growth in primary motoneurons. (A) Western blot analysis of lysates from Hnrnpr+/+ and Hnrnprtm1a/tm1a motoneurons cultured for 7 DIV, probed with an antibody against the C-terminus of hnRNP R. Gapdh was used as loading control. (B) Representative images of primary motoneurons cultured for 7 DIV and immunostained with antibodies against the C-terminal or N-terminal domain of hnRNP R, and with an antibody against β-Tubulin. Scale bars: 5 μm. (C) Quantification of mean fluorescence intensity of the hnRNP R immunosignal obtained with the C-terminal-specific antibody in motoneurons shown in (B). Data are mean ± SD (n = 3 independent experiments; N = 30 motoneurons each for Hnrnpr+/+ and Hnrnprtm1a/tm1a). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; n.s., not significant. (D) Quantification of the survival of motoneurons at 7 DIV as percentage of the number of motoneurons at 1 DIV, in the presence or absence of BDNF. Data are mean ± SD (n = 3 independent experiments; motoneurons were from N = 4 embryos each for Hnrnpr+/+ and Hnrnprtm1a/tm1a). Statistical analysis was performed using…

Figure 5.. Increased DNA damage and impaired DDR in Hnrnprtm1a/tm1a motoneurons. (A) Western blot analysis of subcellular fractions from control motoneurons (0 Gy) or motoneurons subjected to γ-irradiation (9 Gy). Motoneurons were fractioned into cytosolic (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions. Fractions were probed with the indicated antibodies. (B) Western blot analysis of chromatin fractions of NSC-34 cells incubated with Benzonase (Bn) or the indicated amounts of RNase A and separated into supernatant and pellet by centrifugation. (C) Immunoprecipitation of γ-H2AX from nuclear fractions of control (0 Gy) or irradiated (9 Gy) NSC-34 cells. Proteins were analyzed by western blot using antibodies against γ-H2AX or hnRNP R. In, input; IP, immunoprecipitation. (D) Representative images of alkaline comet assays performed on Hnrnpr+/+ and Hnrnprtm1a/tm1a motoneurons cultured for 6 DIV under control conditions (0 Gy) and after exposure to γ-irradiation (9 Gy). (E) Quantification of comet mean tail moments. Data are mean ± SD (n = 3 independent experiments; N = 71 nuclei for Hnrnpr+/+ (0 Gy), N = 55 nuclei for Hnrnpr+/+ (9 Gy), N = 82 nuclei for Hnrnprtm1a/tm1a (0 Gy)…

Figure 6.. Yb1 shows preferential interaction with the full-length isoform of hnRNP R. (A, B) Volcano plots showing proteins identified by mass spectrometry that were significantly enriched after immunoprecipitation with a C-terminal-specific hnRNP R antibody relative to IgG control immunoprecipitations. Experiments were carried out in the absence (A) or presence (B) of Benzonase. Log-transformed p-values (t test) associated with individual proteins were plotted against log-transformed fold changes in protein enrichment between hnRNP R and control immunoprecipitations. (C) Anti-HA immunoprecipitation from HEK293TN cells transfected with plasmids for expression of HA-tagged hnRNP R isoforms or deletion mutants, or HA-tagged EGFP, or from untransfected cells (control). Proteins were analyzed by western blot with antibodies against Yb1 and HA-tag. In, input; IP, immunoprecipitation. Input represents 5% of the lysate used for immunoprecipitation. (D) Quantification of Yb1 co-immunoprecipitation with hnRNP R constructs. Data are mean ± SD (n = 5 independent experiments). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P ≤ 0.01. (E, F)…

Figure 7.. Yb1 knockdown results in DNA damage defects in motoneurons. (A, B) Immunoprecipitation of Yb1 (A) or γ-H2AX (B) performed on whole lysates of non-irradiated (0 Gy) or irradiated (9 Gy) NSC-34 cells. Proteins were analyzed by western blot with antibodies against γ-H2AX and Yb1. In, input; IP, immunoprecipitation. (C) Western blot analysis of Yb1 levels in cultured motoneurons transduced with control or shRNA-Yb1. Calnexin was used as loading control. (D) Quantification of western blot data shown in (C). Data are mean ± SD (n = 3 independent experiments). Statistical analysis was performed using unpaired t-test; *P ≤ 0.05. (E) Representative images of alkaline comet assay performed on control and shRNA-Yb1-transduced motoneurons under non-irradiated conditions (0 Gy) or after exposure to γ-irradiation (9 Gy). (F) Quantification of comet mean tail moments. Data are mean ± SD (n = 3 independent experiments; N = 31 nuclei for control (0 Gy), N = 34 nuclei for control (9 Gy), N = 33 nuclei for shRNA-Yb1 (0 Gy) and N = 33 nuclei for shRNA-Yb1 (9 Gy)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; *P ≤ 0.05, **P ≤ 0.01. (G) Representative…

Figure 8.. Recruitment of Yb1 to chromatin in response to DNA damage is impaired in the absence of the full-length hnRNP R isoform. (A) Non-irradiated NSC-34 cells (0 Gy) or NSC-34 cells subjected to γ-irradiation (9 Gy) were separated into cytosolic (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions. Fractions were analyzed by western blot with the indicated antibodies. (B) Quantification of western blot data shown in (A). The expression of Yb1 in each fraction is presented as the percentage of total. Data are mean ± SD (n = 3 independent experiments). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; *P ≤ 0.05. (C) Non-irradiated (0 Gy) or irradiated (9 Gy) NSC-34 cells transduced with control lentivirus or lentivirus expressing shRNA-FL for knockdown of full-length hnRNP R were fractionated and analyzed by western blot with the indicated antibodies. (D, E) Quantification of the Yb1 Western blot data shown in (C) for control conditions (0 Gy) (D) and after exposure to γ-irradiation (9 Gy) (E). The expression of Yb1 in each fraction is presented as the percentage of total. Data are mean ± SD (n = 3 independent experiments).…